scholarly journals Interactions between active-site-serine β-lactamases and mechanism-based inactivators: a kinetic study and an overview

1993 ◽  
Vol 295 (3) ◽  
pp. 705-711 ◽  
Author(s):  
A Matagne ◽  
M F Ghuysen ◽  
J M Frère
Keyword(s):  
Kinetic Study ◽  
Clavulanic Acid ◽  
Active Site ◽  
Rate Constants ◽  
Beta Lactamase ◽  
Beta Lactamases ◽  
Class A

The interactions between three class A beta-lactamases and three beta-lactamase inactivators (clavulanic acid, sulbactam and olivanic acid MM13902) were studied. Interestingly, the interaction between the Streptomyces cacaoi beta-lactamase and clavulanate indicated little irreversible inactivation. With sulbactam, irreversible inactivation was found to occur with the three studied enzymes, but no evidence for transiently inactivated adducts was found. Irreversible inactivation of the S. albus G and S. cacaoi enzymes was particularly slow. With olivanate, irreversible inactivation was also observed with the three enzymes, but with the S. cacaoi enzyme, no hydrolysis could be detected. A tentative summary of the results found in the literature is also presented (including 6 beta-halogenopenicillanates), and the general conclusions underline the diversity of the mechanisms and the wide variations of the rate constants observed when class A beta-lactamases interact with beta-lactamase inactivators, in agreement with the behaviours of the same enzymes towards their good and poor substrates.

scholarly journals Sequence analysis and enzyme kinetics of the L2 serine beta-lactamase from Stenotrophomonas maltophilia.

1997 ◽  
Vol 41 (7) ◽  
pp. 1460-1464 ◽  
Author(s):  
T R Walsh ◽  
A P MacGowan ◽  
P M Bennett
Keyword(s):  
Amino Acids ◽  
Sequence Analysis ◽  
Clavulanic Acid ◽  
Active Site ◽  
Stenotrophomonas Maltophilia ◽  
Leader Peptide ◽  
Beta Lactamase ◽  
Class A ◽  
Cloned Gene ◽  
Kinetics Of

The L2 serine active-site beta-lactamase from Stenotrophomonas maltophilia has been classified as a clavulanic acid-sensitive cephalosporinase. The gene encoding this enzyme from S. maltophilia 1275 IID has been cloned on a 3.3-kb fragment into pK18 under the control of a Ptac promoter to generate recombinant plasmid pUB5840; when expressed in Escherichia coli, this gene confers resistance to cephalosporins and penicillins. Sequence analysis has revealed an open reading frame (ORF) of 909 bp with a GC content of 71.6%, comparable to that of the L1 metallo-beta-lactamase gene (68.4%) from the same bacterium. The ORF encodes an unmodified protein of 303 amino acids with a predicted molecular mass of 31.5 kDa, accommodating a putative leader peptide of 27 amino acids. Comparison of the amino acid sequence with those of other beta-lactamases showed it to be most closely related (54% identity) to the BLA-A beta-lactamase from Yersinia enterocolitica. Sequence identity is most obvious near the STXK active-site motif and the SDN loop motif common to all serine active-site penicillinases. Sequences outside the conserved regions display low homology with comparable regions of other class A penicillinases. Kinetics of the enzyme from the cloned gene demonstrated an increase in activity with cefotaxime but markedly less activity with imipenem than previously reported. Hence, the S. maltophilia L2 beta-lactamase is an inducible Ambler class A beta-lactamase which would account for the sensitivity to clavulanic acid.

scholarly journals Phosphonate monoester inhibitors of class A β-lactamases

1991 ◽  
Vol 275 (3) ◽  
pp. 793-795 ◽  
Author(s):  
J Rahil ◽  
R F Pratt
Keyword(s):  
Enzyme Inhibition ◽  
Mechanism Of Action ◽  
Active Site ◽  
General Structure ◽  
Beta Lactamase ◽  
Beta Lactamases ◽  
Inhibitory Ability ◽  
Class A ◽  
Structure Formula ◽  
Slow Turnover

Phosphonate monoesters with the general structure: [formula: see text] are inhibitors of representative class A and class C beta-lactamases. This result extends the range of this type of inhibitor to the class A enzymes. Compounds where X is an electron-withdrawing substituent are better inhibitors than the unsubstituted analogue (X = H), and enzyme inhibition is concerted with stoichiometric release of the substituted phenol. Slow turnover of the phosphonates also occurs. These observations support the proposition that the mechanism of action of these inhibitors involves phosphorylation of the beta-lactamase active site. The inhibitory ability of these phosphonates suggests that the beta-lactamase active site is very effective at stabilizing negatively charged transition states. One of the compounds described also inactivated the Streptomyces R61 D-alanyl-D-alanine carboxypeptidase/transpeptidase.

scholarly journals The mutation Lys234His yields a class A β-lactamase with a novel pH-dependence

1991 ◽  
Vol 278 (3) ◽  
pp. 673-678 ◽  
Author(s):  
J Brannigan ◽  
A Matagne ◽  
F Jacob ◽  
C Damblon ◽  
B Joris ◽  
...  
Keyword(s):  
Active Site ◽  
Positive Charge ◽  
Ph Dependence ◽  
Side Chain ◽  
Beta Lactamase ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Beta Lactamases ◽  
Class A ◽  
Transition State Stabilization

The lysine-234 residue is highly conserved in beta-lactamases and in nearly all active-site-serine penicillin-recognizing enzymes. Its replacement by a histidine residue in the Streptomyces albus G class A beta-lactamase yielded an enzyme the pH-dependence of which was characterized by the appearance of a novel pK, which could be attributed to the newly introduced residue. At low pH, the kcat, value for benzylpenicillin was as high as 50% of that of the wild-type enzyme, demonstrating that an efficient active site was maintained. Both kcat. and kcat/Km dramatically decreased above pH 6 but the decrease in kcat./Km could not be attributed to larger Km values. Thus a positive charge on the side chain of residue 234 appears to be more essential for transition-state stabilization than for initial recognition of the substrate ground state.

scholarly journals Chromosome-encoded β-lactamases of Citrobacter diversus. Interaction with β-iodopenicillanate and labelling of the active site

1988 ◽  
Vol 254 (3) ◽  
pp. 891-893 ◽  
Author(s):  
G Amicosante ◽  
A Oratore ◽  
B Joris ◽  
M Galleni ◽  
J M Frère ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Active Site ◽  
Beta Lactamase ◽  
Beta Lactamases ◽  
Class A ◽  
Rate Characteristic ◽  
Trypsin Hydrolysis ◽  
Citrobacter Diversus

Both forms of the chromosome-encoded beta-lactamase of Citrobacter diversus react with beta-iodopenicillanate at a rate characteristic of class A beta-lactamases. The active site of form I was labelled with the same reagent. The sequence of the peptide obtained after trypsin hydrolysis is identical with that of a peptide obtained in a similar manner from the chromosome-encoded beta-lactamase of Klebsiella pneumoniae.

scholarly journals Structural and kinetic studies on β-lactamase K1 from Klebsiella aerogenes

1986 ◽  
Vol 234 (2) ◽  
pp. 343-347 ◽  
Author(s):  
E L Emanuel ◽  
J Gagnon ◽  
S G Waley
Keyword(s):  
Active Site ◽  
Ph Dependence ◽  
Kinetic Studies ◽  
Thiol Group ◽  
Klebsiella Aerogenes ◽  
Beta Lactamase ◽  
Beta Lactam ◽  
Beta Lactamases ◽  
Class A ◽  
Rate Determining Step

beta-Lactamase K1 from Klebsiella aerogenes 1082E hydrolyses both penicillins and cephalosporins comparably and is inhibited by mercurials but not by cloxacillin. These properties distinguish it from those other beta-lactamases that have been allotted to classes on the basis of their amino sequences. beta-Lactamase K1 has been isolated by affinity chromatography; its composition shows resemblances to class A beta-lactamases. Moreover, the N-terminal sequence is similar to those of class A beta-lactamases: there is about 30% identity over the first 32 residues. Furthermore, a putative active-site octapeptide has been isolated and its sequence is similar to the region around the active-site serine residue in class A beta-lactamases. There is one thiol group in beta-lactamase K1; it is not essential for activity. The pH-dependence of kcat. and kcat./Km for the hydrolysis of benzylpenicillin by beta-lactamase K1 were closely similar, suggesting that the rate-determining step is cleavage of the beta-lactam ring.

scholarly journals Effect of the 3′-leaving group on turnover of cephem antibiotics by a class C β-lactamase

1989 ◽  
Vol 259 (1) ◽  
pp. 255-260 ◽  
Author(s):  
L J Mazzella ◽  
R F Pratt
Keyword(s):  
Active Site ◽  
First Generation ◽  
Enterobacter Cloacae ◽  
Beta Lactamase ◽  
Beta Lactamases ◽  
Class A ◽  
Leaving Group ◽  
Enzyme Intermediates ◽  
Class C ◽  
Third Generation Cephalosporins

It has been previously demonstrated for class A beta-lactamases and the DD-peptidase of Streptomyces R61 that the presence of a leaving group at the 3′-position of a cephalosporin can lead to the generation of more-inert acyl-enzyme intermediates than from cephalosporins lacking such a leaving group, and thus to beta-lactamase inhibitors and potentially better antibiotics. In the present work we extend this result to a class C beta-lactamase, that of Enterobacter cloacae P99. The effect is not seen with first-generation cephalosporins, since here deacylation generally seems faster than elimination of the leaving group, but it does clearly appear with cephamycins and third-generation cephalosporins. The structural and/or mechanistic features of the active site giving rise to this phenomenon may thus be common to all serine beta-lactamases and transpeptidases.

scholarly journals A comparative study of class-D β-lactamases

1993 ◽  
Vol 292 (2) ◽  
pp. 555-562 ◽  
Author(s):  
P Ledent ◽  
X Raquet ◽  
B Joris ◽  
J Van Beeumen ◽  
J M Frère
Keyword(s):  
Active Site ◽  
Gene Sequence ◽  
Catalytic Properties ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Beta Lactamase ◽  
Serine Residue ◽  
Beta Lactamases ◽  
Class D ◽  
Burst Kinetics

Three class-D beta-lactamases (OXA2, OXA1 and PSE2) were produced and purified to protein homogeneity. 6 beta-Iodopenicillanate inactivated the OXA2 enzyme without detectable turnover. Labelling of the same beta-lactamase with 6 beta-iodo[3H]penicillanate allowed the identification of Ser-70 as the active-site serine residue. In agreement with previous reports, the apparent M(r) of the OXA2 enzyme as determined by molecular-sieve filtration, was significantly higher than that deduced from the gene sequence, but this was not due to an equilibrium between a monomer and a dimer. The heterogeneity of the OXA2 beta-lactamase on ion-exchange chromatography contrasted with the similarity of the catalytic properties of the various forms. A first overview of the enzymic properties of the three ‘oxacillinases’ is presented. With the OXA2 enzyme, ‘burst’ kinetics, implying branched pathways, seemed to prevail with many substrates.

scholarly journals Ragged N-termini and other variants of class A β-lactamases analysed by chromatofocusing

1991 ◽  
Vol 273 (3) ◽  
pp. 503-510 ◽  
Author(s):  
A Matagne ◽  
B Joris ◽  
J Van Beeumen ◽  
J M Frère
Keyword(s):  
Experimental Error ◽  
Catalytic Properties ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Beta Lactamase ◽  
Beta Lactamases ◽  
Gram Positive Bacteria ◽  
Class A ◽  
Partial Inactivation ◽  
Asparagine Residue

Four beta-lactamases excreted by Gram-positive bacteria exhibited microheterogeneity when analysed by chromatofocusing or ion-exchange chromatography. Ragged N-termini were in part responsible for the charge variants, but deamidation of an asparagine residue was also involved, at least for the Bacillus licheniformis enzyme. The activity of a contaminating proteinase could also be demonstrated in the case of Actinomadura R39 beta-lactamase. With that enzyme, proteolysis resulted in partial inactivation, but the inactivated fragments were easily separated from the active forms. With these, as with the other enzymes, the kinetic parameters of the major variants were identical with those of the mixture within the limits of experimental error, so that the catalytic properties of these enzymes can be determined with the ‘heterogeneous’ preparations.

scholarly journals Characterization of beta-lactamase gene blaPER-2, which encodes an extended-spectrum class A beta-lactamase.

1996 ◽  
Vol 40 (3) ◽  
pp. 616-620 ◽  
Author(s):  
A Bauernfeind ◽  
I Stemplinger ◽  
R Jungwirth ◽  
P Mangold ◽  
S Amann ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Beta Lactamase ◽  
Reading Frame ◽  
Beta Lactamases ◽  
Class A ◽  
Extended Spectrum ◽  
The Family ◽  
Extended Spectrum Beta Lactamases ◽  
Lactamase Gene

Plasmidic extended-spectrum beta-lactamases of Ambler class A are mostly inactive against ceftibuten. Salmonella typhimurium JMC isolated in Argentina harbors a bla gene located on a plasmid (pMVP-5) which confers transferable resistance to oxyiminocephalosporins, aztreonam, and ceftibuten. The beta-lactamase PER-2 (formerly ceftibutenase-1; CTI-1) is highly susceptible to inhibition by clavulanate and is located at a pI of 5.4 after isoelectric focusing. The blaPER-2 gene was cloned and sequenced. The nucleotide sequence of a 2.2-kb insert in vector pBluescript includes an open reading frame of 927 bp. Comparison of the deduced amino acid sequence of PER-2 with those of other beta-lactamases indicates that PER-2 is not closely related to TEM or SHV enzymes (25 to 26% homology). PER-2 is most closely related to PER-1 (86.4% homology), an Ambler class A enzyme first detected in Pseudomonas aeruginosa. An enzyme with an amino acid sequence identical to that of PER-1, meanwhile, was found in various members of the family Enterobacteriaceae isolated from patients in Turkey. Our data indicate that PER-2 and PER-1 represent a new group of Ambler class A extended-spectrum beta-lactamases. PER-2 so far has been detected only in pathogens (S. typhimurium, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis) isolated from patients in South America, while the incidence of PER-1-producing strains so far has been restricted to Turkey, where it occurs both in members of the family Enterobacteriaceae and in P. aeruginosa.

scholarly journals Properties of IRT-14 (TEM-45), a newly characterized mutant of TEM-type beta-lactamases.

1997 ◽  
Vol 41 (2) ◽  
pp. 374-378 ◽  
Author(s):  
M M Caniça ◽  
M Barthélémy ◽  
L Gilly ◽  
R Labia ◽  
R Krishnamoorthy ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Kinetic Parameters ◽  
Clavulanic Acid ◽  
Broad Spectrum ◽  
Promoter Region ◽  
Beta Lactamase ◽  
Beta Lactamases ◽  
Structural Modifications ◽  
E Coli

IRT-14 (TEM-45) is a new mutant TEM-type beta-lactamase that was isolated from clinical Escherichia coli P37 and that confers resistance to broad-spectrum penicillins with reduced sensitivity to beta-lactamase inhibitors. The MICs of amoxicillin alone and of amoxicillin combined with 2 micrograms of clavulanic acid or 2 micrograms of tazobactam per ml were 4,096, 2,048, and 1,024 micrograms/ml, respectively. The strain was susceptible to cephalosporins, aztreonam, moxalactam, and imipenem. The enzyme was purified to homogeneity, and values of the kinetic parameters Kcat, Km, and Kcat/Km were determined for different substrates. This enzyme, with a pI of 5.2, was found to have reduced affinity for broad-spectrum penicillins and cephalosporins. The values of 50% inhibitory concentrations of clavulanic acid, sulbactam, tazobactam, and brobactam are correlated with the higher KmS for substrates. The resistance of E. coli P37 to mechanism-based inactivators results from a higher level of production of the TEM-derived enzyme due to the G-to-T substitution at position 162 (G-162-->T) in the promoter region of blaTEM and from the structural modifications resulting from the Met-69-->Leu and Arg-275-->Gln substitutions that characterize IRT-14 beta-lactamase.

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